Imaging of submicron objects with the light microscope

Abstract

Using conventional optics coupled with image processing, several submicron objects have been studied with light microscopy. These include polystyrene beads (88, 264 and 557 nm), frustules from the diatom Pleurosigma angulatum and the T‐4 bacteriophage, either attached to its host, Escherichia coli, or free in the medium. The best results were obtained from dark‐field and phase contrast optics. Digital image processing with the use of colour look‐up tables in real time greatly promoted precise focusing of the objects. Selective discrimination of image histogram grey values allowed for selection of a sharp contour boundary, thus more effectively delimiting the submicron objects. User interactive scaling of the diffraction limited boundaries greatly improved discrimination of the objects. While Abbe limits have not been surpassed by these techniques, the greater ‘apparent resolution’ could be attributable to a ‘better recognition’ of the submicron objects examined. Equivalence of images of polystyrene beads and bacteriophages was demonstrated with light and electron microscopy of the same field.